Liposomal simvastatin inhibits tumor growth via targeting tumor-associated macrophages-mediated oxidative stress

Cancer Letters 356 (2015) 946–952

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

Liposomal simvastatin inhibits tumor growth via targeting tumorassociated macrophages-mediated oxidative stress Marius Costel Alupei a,b, Emilia Licarete b, Laura Patras a,b, Manuela Banciu a,b,* a b

Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, Babes-Bolyai University, Cluj-Napoca, Romania Molecular Biology Centre, Institute for Interdisciplinary Research in Bio-Nano-Sciences, Babes-Bolyai University, Cluj-Napoca, Romania

A R T I C L E

I N F O

Article history: Received 3 October 2014 Received in revised form 5 November 2014 Accepted 5 November 2014 Keywords: Lipophilic statin Liposomes Cancer Tumor-associated macrophages Oxidative stress

A B S T R A C T

Statins possess antitumor actions at doses 100- to 500-fold higher than those needed to lower cholesterol levels. Thus, the antitumor efficacy of statins could be improved greatly by using tumor-targeted delivery systems. Therefore the present work aims to investigate the antitumor activity of longcirculating liposome-encapsulated simvastatin (LCL-SIM) versus free SIM in B16.F10 murine melanomabearing mice. Our results showed that LCL-SIM inhibits strongly the B16.F10 melanoma growth (by 85%) whereas free SIM was ineffective. Moreover, the antitumor activity of LCL-SIM depends on the presence of functional tumor-associated macrophages (TAM) in tumor tissue and is mainly based on the reduction of the TAM-mediated oxidative stress as well as of the production of the hypoxia-inducible factor 1 α (HIF-1 α) in tumors. In conclusion, our findings suggest that the antitumor activity of LCL-SIM on B16.F10 melanoma growth is a result of the tumor-targeting property of the liposome formulation and is tightly dependent on the presence of TAM in tumor tissue. © 2014 Published by Elsevier Ireland Ltd.

Introduction Apart from cholesterol lowering activity, statins, at very high doses, possess antitumor activity due to their pleiotropic actions on key regulatory molecules (small GTP-binding proteins such as Rho, Rac, or Ras) of intracellular signaling pathways responsible for cell proliferation, apoptosis, inflammation, angiogenesis, and oxidative stress [1–7]. Therefore, tumor-specific delivery of statins is an attractive strategy to increase intratumor drug concentrations, also limit their side effects on healthy tissues and thereby, intensify the therapeutic index of these pharmacological agents. In the present study, we take advantage of long-circulating liposomes (LCLs) to efficiently deliver simvastatin (SIM) into B16.F10 murine melanoma tumors. It is known that LCLs have the capacity to extravasate through the hyperpermeable vasculature of the tumors and to accumulate in the malignant tissue [8]. Moreover, it has been previously proven that LCLs have a natural tropism for tumor-associated macrophages (TAMs) [9,10]. Thus, all processes favorable for tumor development and coordinated by TAM [9] might be significantly affected by properly designed LCL-encapsulated statins. Therefore, in the present study the antitumor activity of LCL incorporating SIM

* Corresponding author. Tel.: +0040264431691; fax: +0040264431858. E-mail address: [email protected] (M. Banciu). http://dx.doi.org/10.1016/j.canlet.2014.11.010 0304-3835/© 2014 Published by Elsevier Ireland Ltd.

(LCL-SIM) and the role of TAM in the antitumor activity of LCLSIM were investigated in the B16.F10 melanoma model. Materials and methods Preparation of LCL-SIM LCL-SIM was prepared by using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (Lipoid GmbH, Ludwigshafen, Germany), polyethylene glycol 2000distearoylphosphatidylethanolamine (PEG-2000-DSPE) (Lipoid GmbH, Ludwigshafen, Germany), cholesterol (Sigma, St. Louis, USA) and SIM (Sigma, St. Louis, USA in a molar ratio of 17:1.011:1:1.209. Nano-sized LCLs were obtained via lipid film hydration method followed by multiple extrusion steps as described previously [10,11]. Unencapsulated drug and lipid aggregates were removed by centrifugation at 15,000 × g at 4 °C for 15 minutes, and the supernatant containing liposomes was collected for further use. LCL-SIM was characterized regarding particle size distribution, zeta potential, and encapsulation efficiency as described previously [12,13]. In this study, to deliver SIM passively to tumor tissue, LCLs with mean particle size around 100 nm and a polydispersity value lower than 0.1 were prepared. The low polydispersity values obtained indicate a narrow size distribution. The ζ potential of liposomes was about 0.1 mV indicating a near neutral surface charge. The encapsulation efficiency was about 33% (33.33 μg SIM/μmole of phospholipid).

Preparation of Lip-CLOD Lip-CLOD consists of a mixture of two types of clodronate-encapsulating liposomes in a ratio of 1:1 (w/w). To deplete TAM, we prepared clodronate-containing LCLs by using film hydration method as described previously [9] with a diameter around 100 nm and polydispersity lower than 0.1. To prevent chemoattraction of systemic monocytes from the bloodstream in tumors, clodronate-containing large

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negatively charged liposomes with the final size around 1 μm were used and prepared as shown by Banciu et al. [9].

was about 6 min. Data were expressed as μmoles of MDA/g of tumor. Each sample was determined in triplicate.

Cells

Determination of nitric oxide metabolites

B16.F10 murine melanoma cells (ATCC, CRL-6475) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Lonza), supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B, 4 mM L-glutamine and 5% NaHCO3 (Lonza) as monolayer at 37 °C in a 5% CO2 humidified atmosphere.

To assess whether the treatments applied can affect tumor production of nitric oxide (NO) – a key oxidative stress marker, the levels of nitrites as stable final products of NO metabolism [20] were determined in tumor lysates. For this purpose, after chemical interaction between Griess reagent [21] and nitrites, a highly colored azo dye was formed and determined at 548 nm. Data were expressed as nmoles of nitrites/g of tumor. Each sample was determined in duplicate.

Murine tumor model Determination of catalytic activity of catalase Male C57Bl/6 mice (6–8 weeks of age) were obtained from Charles River (Hungary) and kept in standard housing with standard rodent chow and water available ad libitum, and a 12-hour light/dark cycle. Experiments were performed according to the national regulations and were approved by the local animal experiments ethical committee (registration no.31054/19.06.2012). For tumor induction, 106 B16.F10 cells were inoculated subcutaneously (s.c.) in the right flank of mice. The B16.F10 tumors became palpable at day 10 after cell inoculation. Body weight of mice was monitored regularly during treatments. After mice sacrification, serum cholesterol levels were determined spectrophotometrically using the Liebermann–Burchard reaction [14]. No significant differences in serum cholesterol levels were observed after any treatment. Additionally, no variation in body mass during the treatment period was encountered. Effects of LCL-SIM on tumor growth LCL-SIM was administered i.v. (in caudal vein) at a dose of 5 mg/kg on days 11 and 14 after tumor cell inoculation. This dosing schedule was selected based on previous studies on the effects of other type of statin (pravastatin) encapsulated in LCL on tumor growth [15]. The same dose of free SIM solubilized in 10% dimethylsulfoxide (DMSO) (Sigma-Aldrich, USA) was administered i.v. at the same time points. As control tumors, tumors from mice treated with PBS were used. Five animals were used per experimental group. Tumor size was measured regularly, and tumor volume was determined according to the formula: V = 0.52a2b, where a is the smallest and b is the largest superficial diameter. On day 15, mice were sacrificed and tumors were measured and collected.

The ability of tumor cells to defend against peroxide radicals was assessed by determination of the catalytic activity of catalase according to Aebi [22]. The activity of catalase for each tumor lysate group was expressed as units of catalytic activity/ mg of total protein and measured in triplicate. Assessment of HIF-1α production To determine the effects of different treatments on the intratumoral levels of HIF-1α-a key transcription factor for tumor development, Western blot analysis was performed. 80 μg of total protein in each tumor tissue lysate was loaded per lane onto a 7.5% polyacrylamide gel. Electrophoresis and Western blot analysis steps were performed as described by Alupei et al. [16]. The amount of HIF-1α in each experimental group was compared to that recorded in control tumors (tumors from mice treated with PBS). The final results represent mean ± SD of three independent experiments. Statistical analysis Data from different experiments were reported as mean ± SD. Statistical comparisons of the overall effects of different treatments on tumors were evaluated by one-way ANOVA with Dunnett’s post-test for multiple comparisons. The differences between the effects of different treatments on angiogenic/inflammatory factor production were analyzed by two-way ANOVA with Bonferroni correction for multiple comparisons using GraphPad Prism version 6 for Windows, GraphPad Software (San Diego, CA). A value of P < 0.05 was considered significant.

Effects of lip-CLOD pretreatment on the antitumor activity of LCL-SIM

Results To determine whether TAM play an important role in the antitumor activity of LCL-SIM, B16.F10 melanoma-bearing mice were pretreated 24 h before administration of LCL-SIM with 25 mg/kg of Lip-CLOD (at day 10 after tumor cell inoculation) injected i.v.. This dosing schedule was selected based on previous studies on the TAMdepleting actions of Lip-CLOD in B16.F10 tumors [9]. As controls, tumor-bearing mice treated with PBS which did not receive Lip-CLOD treatment were used. Four to five animals were used per experimental group. On day 15, mice were sacrificed and tumor volumes were measured and collected. Assessment of tumor production of proteins involved in tumor angiogenesis and inflammation After mice sacrification tumors were isolated, weighed, and pooled tumor tissue lysates for each group were obtained as shown by Alupei et al., in 2014 [16]. Then, the protein content of the tissue lysates was determined by biuret method [17]. To evaluate the effects of different treatments on angiogenic/inflammatory protein production in tumor tissue a screening by using a protein array of RayBio® Mouse Angiogenic protein Antibody Array membranes 1.1 (RayBiotech Inc., Norcross, GA, USA) was performed as described previously [18]. The protein expression level was quantified by measuring the intensity of the color of each spot on the membranes, in comparison to the positive control spots already bound to the membranes, using TotalLab Quant Software version 12 for Windows. Each protein level from each experimental group was determined in duplicate and expressed as percentage of the same protein level from the control tumors (untreated tumors). The final results represent mean ± SD of two independent experiments. Determination of malondialdehyde levels To investigate whether different treatments can affect oxidative stress in tumors we performed tumor lysate quantification of malondialdehyde (MDA) – a marker for membrane lipid peroxidation, through high-performance liquid chromatography (HPLC) [19]. Before HPLC analysis of MDA sample deproteinization was performed as described previously [19]. Then samples were centrifuged at 4500 × g for 5 min and 100 μl of each supernatant was used for HPLC analysis. The column type was RP18 (5 μm) (Supelco, Bellefonte, PA, USA) and the mobile phase consisted of 30 mM KH2PO4/methanol in a volume ratio of 65:35. Flow rate was set at 0.5 ml/min and MDA was measured using a UV detector set at 254 nm. The retention time of MDA

Antitumor activity of LCL-SIM versus free SIM on B16.F10 murine melanoma model To compare the effects of LCL-SIM and free SIM on B16.F10 melanoma growth, mice received two i.v. injections of 5 mg/kg of SIM of either formulation at the moment the tumor had a diameter of approximately 4 mm (day 11) and at day 14 after tumor cell inoculation. The antitumor activities induced by LCL-SIM and free SIM were analyzed by determining the area under the tumor growth curves (AUTC) until day 15 (the day when mice were sacrificed) (Fig. 1A) as well as inhibition of tumor growth compared to control tumors (tumors in mice treated with PBS) at day of sacrification (Fig. 1B). Tumor growth was strongly decelerated after LCL-SIM treatment when compared to control tumors (P < 0.05) while administration of free SIM did not exert any inhibition of tumor growth (Fig. 1A). In line with AUTC data, tumor volumes were significantly smaller for LCL-SIM as compared to free drug or PBStreated mice (P < 0.01, Fig. 1B).The strong inhibition of tumor growth at day 15 induced by LCL-SIM (by 85% compared to control group, Fig. 1 panel B) is clearly enabled by the tumor-targeting property of the liposome formulation since SIM administered as free form at the same dose did not show any inhibitory effect. Antitumor activity of LCL-SIM depends on the presence of TAM in B16.F10 melanoma tumors To determine whether the antitumor activity of LCL-SIM in the B16.F10 melanoma model is dependent on the TAM functions in tumor tissue, tumor-bearing mice were pretreated with 25 mg/kg of Lip-CLOD 24 h before administration of 5 mg/kg of LCL-SIM at

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Fig. 2. Effects of Lip-CLOD pretreatment on the antitumor activity of LCL-SIM. Tumor growth for each experimental group is analyzed by comparing tumor volumes at day 15 after tumor cell inoculation. The results are compared to controls and expressed as mean ± SD of five mice. *, P < 0.05; **, P < 0.01. Control: group that received only PBS at days 11 and 14 after tumor cell inoculation; Lip-CLOD: group that received only 25 mg/kg of Lip-CLOD at day 10 after tumor cell inoculation; LipCLOD + LCL-SIM: group that received Lip-CLOD at day 10 after tumor cell inoculation followed by treatment with 5 mg/kg of LCL-SIM administered at days 11 and 14 after tumor cell inoculation.

800 600 400

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reduced only slightly the levels of the majority of intratumoral proangiogenic/proinflammatory factors (by 10%, P < 0.01) except for leptin that was moderately reduced by 35% compared to its control levels (Table 1). The levels of antiangiogenic/antiinflammatory proteins were not statistically significantly affected by LCL-SIM without pretreatment with Lip-CLOD (Table 1). When Lip-CLOD was given, LCL-SIM exerted moderate inhibitory effects on the production of

0 Control

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LCL-SIM

Fig. 1. Antitumor activity of LCL-SIM versus free SIM in B16.F10 murine melanoma models. A. AUTCs after different treatments; B. Tumor volumes at day 15 after different treatments. The results are compared to PBS-treated groups (controls) and expressed as mean ± SD of five mice. AUTC, area under the tumor growth curve; ns, not significant (P > 0.05); **, P < 0.01; Control: group that received only PBS at days 11 and 14 after tumor cell inoculation; Free SIM: group that received 5 mg/kg of SIM as free form at days 11 and 14 after tumor cell inoculation; LCL-SIM: group that received 5 mg/kg of SIM as liposomal formulation at days 11 and 14 after tumor cell inoculation.

days 11 and 14 after tumor cell inoculation. The effects of LipCLOD pretreatment on the antitumor activity of LCL-SIM were analyzed using tumor volumes at day 15 (Fig. 2). In line with previous results [9] the growth of tumors only pretreated with LipCLOD (not followed by other treatments) was inhibited by 55% (P < 0.01) compared with control tumors (tumors from mice that received only PBS) (Fig. 2). Interestingly when Lip-CLOD pretreatment was administered no additional inhibitory effect of LCL-SIM on tumor growth was seen (Fig. 2). It is likely that the antitumor effect of LCL-SIM treatment administered in the Lip-CLOD–pretreated group was annihilated by TAM depletion. Effect of LCL-SIM treatments on tumor angiogenesis As shown previously [9] the average reduction of most of the angiogenic/inflammatory protein levels in tumors that received only Lip-CLOD was about 55% (P < 0.001) compared to the same reduction in control tumors (Table 1). Free SIM administration did not affect the levels of angiogenic/inflammatory proteins (data not shown). When Lip-CLOD pretreatment was not administered, LCL-SIM

Table 1 Effects of i.v. administered LCL-SIM and Lip-CLOD on angiogenic/inflammatory factor production. Angiogenic/ Percentage of inhibition (−) and stimulation (+) of inflammatory intratumor production of proteins involved in tumor angiogenesis/inflammation after different treatments factors Lip-CLOD G-CSF GM-CSF M-CSF IGF-II IL-1α IL-1β IL-6 IL-9 IL-12p40 IL-13 TNF-α MCP1 Eotaxin FasL bFGF VEGF Leptin TPO TIMP-1 TIMP-2 PF-4 IL-12p70 IFN-γ MIG

LCL-SIM

−74.49 ± 0.39 (***) −18.34 ± 2.01 (ns) −59.58 ± 3.38 (***) −10.036 ± 3.32 (ns) −39.86 ± 3.76 (***) −1.67 ± 5.93 (ns) −7.36 ± 5.46 (ns) −46.90 ± 3.03 (***) −20.77 ± 1.56 (ns) −21.81 ± 6.24 (ns) −73.82 ± 5.36 (***) −16.54 ± 6.61(ns) −70.07 ± 2.04 (***) −12.59 ± 9.33 (ns) −40.82 ± 3.34 (***) +2.18 ± 6.31 (ns) −64.48 ± 5.44 (***) −23.01 ± 2.41 (ns) −48.48 ± 2.88 (***) −0.33 ± 2.64 (ns) −58.08 ± 4.21 (***) −0.01 ± 0.78 (ns) −34.77 ± 0.58 (**) −23.19 ± 1.11 (ns) −85.26 ± 4.29 (***) −1.02 ± 4.92 (ns) −76.97 ± 3.44 (***) +1.59 ± 3.25 (ns) −55.27 ± 2.62 (***) +3.23 ± 4.04 (ns) −36.87 ± 4.58 (***) −19.36 ± 3.86 (ns) −93.61 ± 4.76 (***) −34.90 ± 1.94 (**) −36.09 ± 2.18 (**) −3.52 ± 18.29 (ns) −57.02 ± 1.11 (***) +7.04 ± 4.21 (ns) −53.70 ± 4.76 (***) +3.52 ± 5.74 (ns) −42.50 ± 10.64 (***) +2.19 ± 10.37 (ns) −35.39 ± 0.54 (**) +3.27 ± 11.91 (ns) −73.20 ± 2.17 (***) −6.39 ± 3.81 (ns) (ns) −74.91 ± 3.63 (***) −16.51 ± 5.09

Lip-CLOD + LCL-SIM −20.43 ± 1.78 (ns) −6.32 ± 4.20 (ns) −36.19 ± 7.95 (**) −5.08 ± 6.70 (ns) −2.75 ± 7.09 (ns) −54.35 ± 1.48 (***) −34.74 ± 1.31 (**) −22.15 ± 1.17 (ns) −23.46 ± 2.99 (ns) −23.39 ± 2.14 (ns) −33.79 ± 0.01 (**) −25.84 ± 3.97 (ns) −65.47 ± 10.26 (***) −42.71 ± 5.21 (***) −14.94 ± 8.19 (ns) −37.05 ± 0.01 (***) −68.99 ± 5.38 (***) −21.39 ± 0.01 (ns) +7.86 ± 0.01 (ns) −48.92 ± 0.01 (***) −23.33 ± 0.98 (ns) −19.25 ± 7.23 (ns) −53.16 ± 0.06 (***) −73.39 ± 5.44 (***)

The protein levels are compared to protein levels in control tumors which were treated only with PBS. The results represent the mean ± SD of two independent measurements. ns, not significant (P > 0.05). ** , P < 0.01. *** , P < 0.001.

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Fig. 3. Tumor MDA levels after different treatments. A. MDA levels in tumors from mice that did not receive Lip-CLOD pretreatment. B. MDA levels in tumors pretreated with Lip-CLOD. The results are compared to control levels and expressed as mean ± SD of three independent measurements. ns, not significant (P > 0.05); ***, P < 0.001; Control: group that received only PBS at days 11 and 14 after tumor cell inoculation; Free SIM: group that received 5 mg/kg of SIM as free form at days 11 and 14 after tumor cell inoculation; LCL-SIM: group that received 5 mg/kg of SIM as liposomal formulation at days 11 and 14 after tumor cell inoculation; Lip-CLOD: group that received only 25 mg/ kg of Lip-CLOD at day 10 after tumor cell inoculation; Lip-CLOD + LCL-SIM: group that received Lip-CLOD at day 10 after tumor cell inoculation followed by treatment with 5 mg/kg of LCL-SIM administered at days 11 and 14 after tumor cell inoculation.

of NO metabolism [23]. Nitrites levels were two times lower compared to the control levels after treatment with LCL-SIM applied in the group which did not receive Lip-CLOD (Fig. 4A). Free SIM administration did not affect the production of nitrites in tumors (Fig. 4A). Moreover, Lip-CLOD alone also reduced levels of nitrite (by 36%), suggesting the important role of TAM in the production of NO (Fig. 4B). When Lip-CLOD administration was followed by LCLSIM treatment nitrite production in tumors was similar to the nitrite production in control tumors but higher (by 50%, P < 0.05) than in tumors from mice that received only Lip-CLOD (Fig. 4B). To investigate whether LCL-SIM can affect enzymatic antioxidant defense mechanisms in B16.F10 melanoma model in vivo, the effects of LCL-SIM with and without Lip-CLOD pretreatment were studied by determination of the catalytic activities of catalase (Fig. 5A and B). LCL-SIM administered in tumors with infiltrated TAM, reduced strongly (by 50%, P < 0.05) the catalytic activity of catalase compared to the activity of the same enzyme in control tumors (Fig. 5A and B). Administration of LCL-SIM after TAM depletion did not affect the catalytic activity of catalase (Fig. 5B). Interestingly, tumor treated with Lip-CLOD alone showed increased catalase activity by 50% (P < 0.05) compared to control catalytic activities of this enzyme (Fig. 5A and B).

proteins involved in tumor angiogenesis/inflammation (average reduction of 32% compared to controls) (P < 0.001). It seems that reduction of the production of the angiogenic/inflammatory proteins determined by TAM depletion was attenuated when LipCLOD pretreament was followed by LCL-SIM administration (Table 1). Involvement of oxidative stress in the antitumor activity of LCL-SIM treatment

A nmoles of nitrites/g of tumor

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nmoles of nitrites/g of tumor

To determine whether the antitumor activity of LCL-SIM is related to modulatory actions on oxidative stress, we quantified important oxidative stress markers in tumor tissue lysates such as MDA, nitrites, and catalytic activity of catalase. The results are shown in Figs. 3–5. When Lip-CLOD was not given MDA levels in tumors treated with LCL-SIM were two times lower than in control tumors while MDA levels in free SIM-treated tumor were similar to those noted in controls (Fig. 3A). When mice received Lip-CLOD pretreatment, tumor levels of MDA were not affected by any treatment (Fig. 3B). To assess whether the effects of NO production in tumors play an important role in the antitumor activity of LCL-SIM we determined intratumoral production of nitrites, the stable final products

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Fig. 4. Tumor nitrite levels after different treatments. A. Nitrite levels in tumors from mice that did not receive Lip-CLOD pretreatment. B. Nitrite levels in tumors pretreated with Lip-CLOD. The results are compared to control levels and expressed as mean ± SD of two independent measurements. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; Control: group that received only PBS at days 11 and 14 after tumor cell inoculation; Free SIM: group that received 5 mg/kg of SIM as free form at days 11 and 14 after tumor cell inoculation; LCL-SIM: group that received 5 mg/kg of SIM as liposomal formulation at days 11 and 14 after tumor cell inoculation; Lip-CLOD: group that received only 25 mg/kg of Lip-CLOD at day 10 after tumor cell inoculation; Lip-CLOD + LCL-SIM: group that received Lip-CLOD at day 10 after tumor cell inoculation followed by treatment with 5 mg/kg of LCL-SIM administered at days 11 and 14 after tumor cell inoculation.

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M.C. Alupei et al./Cancer Letters 356 (2015) 946–952

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Fig. 5. Catalytic activity of catalase after different treatments. A. Catalytic activity of catalase in tumors from mice that did not receive Lip-CLOD pretreatment. B. Catalytic activity of catalase in tumors pretreated with Lip-CLOD. The results are compared to the catalytic activity of the same enzyme in controls and expressed as mean ± SD of three independent measurements. ns, not significant (P > 0.05); *, P < 0.05; Control: group that received only PBS at day 11 and 14 after tumor cell inoculation; Free SIM: group that received 5 mg/kg of SIM as free form at days 11 and 14 after tumor cell inoculation; LCL-SIM: group that received 5 mg/kg of SIM as liposomal formulation at days 11 and 14 after tumor cell inoculation; Lip-CLOD: group that received only 25 mg/kg of Lip-CLOD at day 10 after tumor cell inoculation; Lip-CLOD + LCL-SIM: group that received Lip-CLOD at day 10 after tumor cell inoculation followed by treatment with 5 mg/kg of LCL-SIM administered at days 11 and 14 after tumor cell inoculation.

Discussion

Effects of LCL-SIM treatment on HIF-1α production in B16.F10 melanoma model To determine whether the mechanisms of the antitumor activity exerted by LCL-SIM on B16.F10 melanoma tumors involved inhibitory effects on the expression of HIF-1α, Western blot analysis was performed. Among all treatments tested only LCL-SIM without pretreatment with Lip-CLOD decreased strongly (by 55% compared to controls) the level of HIF-1α (P < 0.05) (Fig. 6A and B). It is noted that production of HIF-1α was increased in both LipCLOD-pretreated groups by 45–60% compared to its levels in control tumors (Fig. 6B) (P < 0.001).

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Statins at 100- to 500-fold higher doses than those applied in the cholesterol-related pathologies have antitumor properties [15,16]. Therefore, tumor-targeted delivery of statins could increase intratumoral drug concentrations while limiting side effects on health tissues. In the present paper, we took the advantage of LCL to target SIM to tumor tissue, and thus we could exploit SIM pleiotropic actions in cancer treatment, which to our knowledge have never been described before. The long-circulation property provides the liposomes with the opportunity to substantially extravasate and accumulate SIM in tumors [24,25]. The tumor-targeting property of

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Fig. 6. HIF-1α production in B16.F10 melanoma tumors after different treatments. A. HIF-1α levels in tumors from mice that did not receive Lip-CLOD pretreatment. B. HIF-1α levels in tumors in tumors pretreated with Lip-CLOD. The results are compared to the HIF-1α levels in control tumors and expressed as mean ± SD of three independent measurements. GAPDH, glyceraldehyde-3-phosphate dehydrogenase as loading control; ns, not significant (P > 0.05); *, P < 0.05; ***, P < 0.001 Control: group that received only PBS at days 11 and 14 after tumor cell inoculation; Free SIM: group that received 5 mg/kg of SIM as free form at days 11 and 14 after tumor cell inoculation; LCL-SIM: group that received 5 mg/kg of SIM as liposomal formulation at days 11 and 14 after tumor cell inoculation; Lip-CLOD: group that received only 25 mg/kg of LipCLOD at day 10 after tumor cell inoculation; Lip-CLOD + LCL-SIM: group that received Lip-CLOD at day 10 after tumor cell inoculation followed by treatment with 5 mg/kg of LCL-SIM administered at days 11 and 14 after tumor cell inoculation.

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LCL is also enabled by the enhanced permeability of tumor vasculature, as compared to healthy endothelium (referred to as “the enhanced permeability and retention” (EPR) effect) [8]. Thus, our data demonstrated that the tumor-targeting property of the LCL enabled the strong inhibition of the growth of B16.F10 tumors exerted by LCL-SIM (by 85% compared to the growth of control tumors) while unencaspulated SIM did not determine any antitumor activity on the same tumor model (Fig. 1A and B). Moreover, previous data regarding intratumoral accumulation of LCL have shown that they accumulated in the endosomal/lysosomal compartment of TAM [10]. This finding proposed TAM as a possible cell type target for the antitumor activity of LCL-SIM. Therefore, to evaluate whether TAM play a crucial role in the antitumor activity of LCL-SIM, we investigated the effects of the Lip-CLOD pretreatment on the antitumor activity of LCL-SIM in B16.F10 melanomabearing mice. Our data suggested clearly that the antitumor activity of LCL-SIM depends on the presence of TAM in tumor tissue as no additional antitumor actions of LCL-SIM on B16.F10 melanoma models were noted when mice were pretreated with Lip-CLOD (Fig. 2). To gain insight on the molecular mechanisms of the dependence between antitumor activity of LCL-SIM and TAM we tested whether this liposomal formulation can affect TAM-mediated processes involved in tumor development such as angiogenesis/ inflammation, oxidative stress as well as metastatic potential of B16.F10 melanoma tumors. Our data suggested that the antitumor activity of LCL-SIM did not involve the inhibition of TAMdriven tumor angiogenesis, since the production of most of the angiogenic/inflammatory proteins produced in TAM was only slightly affected by LCL-SIM administration in mice which were not pretreated with Lip-CLOD (Table 1). Moreover, it seems that LCL-SIM treatment after Lip-CLOD administration diminished the inhibitory effects of TAM depletion on the tumor production of most angiogenic/inflammatory proteins (Table 1). This effect might be related to the proangiogenic actions exerted by SIM [26] when only very low concentrations of this drug in the tumor environment can be achieved as a consequence of the lack of target cell type for LCLSIM in Lip-CLOD-pretreated tumors. These proangiogenic actions were explained previously by the enhancement of endothelial NO synthase activity [26] and can be supported by our results regarding higher intratumor level of nitrites (NO metabolites) in LipCLOD-pretreated tumors that received LCL-SIM compared with their levels in tumors from mice that received only Lip-CLOD (P < 0.05) (Fig. 4B). Since many cancer types including melanoma are under persistent oxidative stress [27,28] and TAM are important contributors in producing the sublethal levels of reactive oxygen species (ROS) and NO in tumor environment [29] our studies investigated the effects of LCL-SIM on oxidative stress required for tumor development in the presence as well as in the absence of TAM. When LipCLOD was not given, only administration of LCL-SIM exerted strong antioxidant activity on B16.F10 melanoma tumors since high decrease of MDA levels (Fig. 3A and B) as well as of production of nitrites (Fig. 4A and B) was noted. Nevertheless, Lip-CLOD alone also induced a moderate reduction of the intratumor production of nitrites (Fig. 4B) suggesting the TAM role in supporting the growth of B16.F10 melanoma through the stimulation of NO production [23]. In addition, catalase activity was enhanced in tumors that received only either LCL-SIM or Lip-CLOD (Fig. 5A and B). This finding might be related to the decrease of the intratumor production of NO since NO was previously described as a reversible inhibitor of this enzyme [30]. Moreover, statin inhibitory effects on the inducible NO synthase expression in macrophages [2,31] can support our data. However, when B16.F10 melanoma-bearing mice were pretreated with Lip-CLOD, no antioxidant effect of LCL-SIM on tumor growth was noted (Figs. 3B and 4B). This is probably due to the opposite effect of low-doses SIM achieved in tumor tissue in the

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absence of target cell type for LCL-SIM. Altogether these data indicate that the antitumor activity of LCL-SIM is likely primarily caused by their suppressive effects on the TAM-mediated oxidative stress in tumors. Furthermore, oxidative stress is also crucial for the progression and the invasion of B16.F10 melanoma tumors via regulation of the expression as well as of the stability of hypoxia inducible factor 1α (HIF-1α) [16,32,33]. Therefore, we gained more evidence about the consequences of the antioxidant activity of LCLSIM on the metastatic potential of B16.F10 melanoma via assessment of the intratumor production of HIF-1α in the presence as well as in the absence of TAM in tumors. Our results showed that only LCLSIM treatment administered alone in mice suppressed strongly (by 55% compared to controls) tumor expression of HIF-1α (Fig. 6A). This beneficial effect might be related to the low levels of ROS insufficient for HIF-1α degradation via suppression of the activity of prolyl hydroxylases in tumors after the same treatment [34]. Moreover, in both Lip-CLOD-pretreated groups the production of HIF-1α was enhanced notably by 45–60% compared to control group (Fig. 6B) probably due to intratumor hypoxia induced by the antiangiogenic effect of the Lip-CLOD pretreatment (Table 1). This observation is consistent with previous reports that described the limited clinical effectiveness of antiangiogenic agents as a consequence of overexpression of HIF-1α in cancer cells [35]. Taken together, the present results point to a strong inhibition of tumor oxidative stress mediated by TAM as the principal cause for the antitumor activity of liposomal SIM in vivo. Consequently, LCL-SIM inhibited strongly intratumor production of HIF-1α, one of the key players in the modulation of the metastatic and survival capacity of B16.F10 melanoma tumors. Nevertheless, the limited antiangiogenic actions of LCL-SIM suggest that future in vivo anticancer therapeutic approaches based on liposomal SIM could be used only in combination with antiangiogenic agents. Acknowledgements This work was financially supported by UEFISCDI (Romanian Ministry of Education, Research and Innovation)-projects (code PN II – RU 387/2010, contract number 145/2010 and code PN-II-PTPCCA-2011-3.2-1060, contract number 95/2012). Conflict of interest statement None. References [1] X.F. Qi, D.H. Kim, Y.S. Yoon, S.K. Kim, D.Q. Cai, Y.C. Teng, et al., Involvement of oxidative stress in simvastatin-induced apoptosis of murine CT26 colon carcinoma cells, Toxicol. Lett. 199 (2010) 277–287. [2] M.I. Yilmaz, Y. Baykal, M. Kilic, A. Sonmez, F. Bulucu, A. Aydin, et al., Effects of statins on oxidative stress, Biol. Trace Elem. Res. 98 (2004) 119–127. [3] M.L. Coleman, M.F. Olson, Rho GTPase signalling pathways in the morphological changes associated with apoptosis, Cell Death Differ. 9 (2002) 493–504. [4] J.K. Liao, U. Laufs, Pleiotropic effects of statins, Annu. Rev. Pharmacol. Toxicol. 45 (2005) 89–118. [5] D. Bar-Sagi, A. Hall, Ras and Rho GTPases: a family reunion, Cell 103 (2000) 227–238. [6] M. Eto, C. Barandier, L. Rathgeb, T. Kozai, H. Joch, Z. Yang, et al., Thrombin suppresses endothelial nitric oxide synthase and upregulates endothelinconverting enzyme-1 expression by distinct pathways: role of Rho/ROCK and mitogen-activated protein kinase, Circ. Res. 89 (2001) 583–590. [7] U. Laufs, J.K. Liao, Direct vascular effects of HMG-CoA reductase inhibitors, Trends Cardiovasc. Med. 10 (2000) 143–148. [8] H. Maeda, T. Sawa, T. Konno, Mechanism of tumor-targeted delivery of macromolecular drugs, including the EPR effect in solid tumor and clinical overview of the prototype polymeric drug SMANCS, J. Control. Release 74 (2001) 47–61. [9] M. Banciu, J.M. Metselaar, R.M. Schiffelers, G. Storm, Antitumor activity of liposomal prednisolone phosphate depends on the presence of functional tumor-associated macrophages in tumor tissue, Neoplasia 10 (2008) 108–117.

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